臺灣博碩士論文加值系統

本研究利用聚合酶鏈鎖反應(PCR)從四株仙人掌桿菌中篩選出BCRC 12811 (經cry gene typing重新鑑定為蘇力菌)及蘇力菌HD-1具有營養期殺蟲蛋白基因vip3，其PCR產物大小約為2.4 kb。將vip3基因與載體pET29b接合構築成表現質體pET29b-vip3-12811及pET29b-vip3-HD1，並分別以轉形方式表現於Escherichia coli BL21宿主中。兩株重組菌株利用IPTG誘導，可分別表現出Vip3-12811及Vip3-HD1兩個蛋白質，分子量大小約為88.5 kDa，由核苷酸序列轉譯後可以推衍出789個胺基酸，經由MS/MS分析，部分胺基酸片段與先前由核苷酸序列推衍之胺基酸序列相吻合。生物檢定結果顯示，轉殖株純化之Vip3-12811毒蛋白對小菜蛾、擬尺蠖及玉米穗蟲1-3齡幼蟲的LC50分別為：91、152、220 μg/mL及256、358、459 μg/mL與279、376、481 μg/mL。而轉殖株純化的Vip3-HD1毒蛋白對小菜蛾、擬尺蠖及玉米穗蟲1-3齡幼蟲的LC50分別為：82、135、188 μg/mL及240、332、415 μg/mL與248、350、432 μg/mL。

Four Bacillus cereus and Bacillus thuringiensis kurstaki HD-1 isolates were screened by polymerase chain reaction (PCR) to detect the presence of vegetative insecticidal protein genes. The vip3 gene was amplified from B. cereus 12811 (reidentified as B. thuringiensis by cry gene typing) and B. thuringiensis HD-1 respectively by PCR using vip28F and vip26R primers. The vip3 gene was cloned into vector pET29b to form construct recombinant expression plasmid pET29b-vip3-12811 and pET29b-vip3-HD1. By transformation, the recombinant expression plasmids were cloned into Escherichia coli BL21 respectively to form recombinant strains of pET29b-vip3-12811 and pET29b-vip3-HD1. The two recombinant strains were induced by IPTG to produce a protein of approximately 88.5 kDa. Moreover, by MS/MS analysis, the nucleotide sequence was determined to predict a protein of 789 amino acid residues. The LC50 of purified recombinant protein (Vip3-12811) against 1st~3rd instar Plutella xylostella, Trichoplusia ni, and Helicoverpa armigera were 91, 152, 220 μg/mL, 279.18, 375.90, 480.93 μg/mL and 256, 358, 459 μg/mL and 279, 376, 481 μg/mL respectively. The LC50 of purified recombinant protein (Vip3-HD1) against 1st~3rd instar Plutella xylostella, Trichoplusia ni, and Helicoverpa armigera were 82, 135, 188 μg/mL, 240, 332, 415 μg/mL and 248, 350, 432 μg/mL respectively.

目 錄

中文摘要................................................................................................................I
英文摘要..............................................................................................................II
目錄.....................................................................................................................IV
圖目錄..............................................................................................................VIII
表目錄..................................................................................................................X
第一章 緒論.........................................................................................................1
1-1 前言……………………………………………………………………1
1-2 研究目的………………………………………………………………2
1-3 研究大綱…….……………………………………………………3
第二章 文獻回顧.................................................................................................4
2-1 蘇力菌之簡介.........................................................................................4
2-1-1 蘇力菌之發現...............................................................................4
2-1-2 蘇力菌之特性..................................................................................4
2-2 仙人掌桿菌之簡介.................................................................................5
2-2-1 仙人掌桿菌生長型態特徵與條件..................................................5
2-2-2 仙人掌桿菌群…………………......................................................5
2-3 營養期殺蟲蛋白之簡介.........................................................................6
2-3-1 VIP之來源.........................................................................................6
2-3-2 VIP與ICP之比較...............................................................................6
2-3-3 VIP之分類.........................................................................................7
2-3-4 VIP之作用機制.................................................................................8
2-3-5 vip3基因選殖與表現......................................................................11
2-3-6 VIP之應用與前景...........................................................................13
第三章 材料與方法...........................................................................................17
3-1 實驗菌種...............................................................................................17
3-2 菌種保存...............................................................................................18
3-3 質體DNA之製備………………..........................................................19
3-4 DNA濃度之測定...................................................................................19
3-5 vip3基因之引子設計.............................................................................20
3-6 聚合酶連鎖反應……...................................................…....................20
3-7 洋菜膠體電泳及染色...........................................................................20
3-8 DNA片段之萃取純化...........................................................................21
3-9 DNA之限制酵素切割反應...................................................................21
3-10 中間質體之構築及轉殖.....................................................................22
3-11 DNA之接合酵素反應.........................................................................22
3-12 勝任細胞之製備.................................................................................23
3-13 表現質體之構築與轉殖大腸桿菌DH5α勝任細胞……...................23
3-14 vip3基因之轉形作用........................................................................24
3-15 E. coli BL21 (pETvip3-12811及pETvip3-HD1)表現蛋白質之誘導..25
3-16 E. coli BL21 (pETvip3-12811及pETvip3-HD1)細胞萃取液之製備..25
3-17 親合性管柱製備及蛋白質純化.........................................................25
3-18 核酸定序.............................................................................................26
3-19 蛋白質之胺基酸片段分析(MS/MS)..................................................27
3-20 總蛋白質之分析.................................................................................27
3-21 十二烷基硫酸鈉-聚丙烯醯胺膠體電泳……...…………………..28
3-22 Coomassie Brilliant blue染色..............................................................29
3-23 西方墨點分析法……………….........................................................29
3-24 野生株初步純化殺蟲蛋白之製備.....................................................30
3-25 生物活性檢定.....................................................................................31
第四章 結果.......................................................................................................32
4-1 篩選含有vip3基因之菌株....................................................................32
4-2 vip3基因中間質體及表現質體之構築..............................................32
4-3 vip基因表現質體轉殖大腸桿菌DH5α勝任細胞與大腸桿BL21 (DE3)受體細胞....................................................................................33
4-4 核酸定序及胺基酸片段分析...............................................................34
4-5 E. coli BL21轉殖株表現蛋白質之分佈與大量誘導表現................35
4-6 親合性管柱純化蛋白質.......................................................................37
4-7 pET29b-vip3-12811及pET29b-vip3-HD1之西方墨點分析..............37
4-8 生物活性檢定.......................................................................................37
第五章 討論.......................................................................................................39
第六章 結論與未來展望...................................................................................44
6-1 結論………….......................................................................................44
6-2 未來展望…….......................................................................................45
圖表集.................................................................................................................46
參考文獻.............................................................................................................67
附錄.....................................................................................................................74

圖 目 錄

圖1. 實驗流程圖................................................................................................46
圖2. 仙人掌桿菌Bacillus cereus BCRC12811及蘇力菌B. thuringiensis kurstaki HD-1之vip3基因與載體pCR-XL- TOPO構築之流程圖……..47
圖3. 仙人掌桿菌Bacillus cereus BCRC12811及蘇力菌B. thuringiensis kurstaki HD-1之vip3基因與載體pET29b構築之流程圖…………........48
圖4. 利用vip28F、vip26R引子組對仙人掌桿菌Bacillus cereus BCRC12811及蘇力菌B. thuringiensis kurstaki HD-1進行PCR以篩選vip3基因片段之1%洋菜膠體電泳分析圖.........................................................................49
圖5. 利用PCR及限制酶剪切確認Bacillus cereus BCRC12811及蘇力菌B. thuringiensis kurstaki HD-1重組質體(pTOP-vip3-12811和pTOPO-vip3-HD1)的vip基因………………………………………....50
圖6. 利用PCR及限制酶剪切確認Bacillus cereus BCRC12811及蘇力菌B. thuringiensis kurstaki HD-1表現質體(pET29b- vip3-12811和pET29b-vip3-HD1)的vip基因..................................................................51
圖7. 重組菌株E. coli BL21 (pET29b-vip3A-12811)的vip3A基因之核苷酸序列與推衍之胺基酸序列………………………………..............................52

圖8. 利用IPTG誘導重組蛋白Vip3 (pETvip3-12811和pETvip3-HD1)之SDS-PAGE分析圖....................................................................................57
圖9. 在不同溫度下，以1 mM的IPTG誘導重組菌株E. coli BL21 (pETvip3- 12811)經不同時間之蛋白質表現圖………………………....................58
圖10. 在16oC下，以1 mM的IPTG誘導重組菌株E. coli BL21 (pETvip3-HD1)經不同時間之蛋白質表現圖…...............................................................59
圖11. 利用1 mM的IPTG於不同時數下誘導重組蛋白質表現之SDS- PAGE分析圖….......................................................................................................60
圖12. 以親合性管柱純化重組菌株E. coli BL21 (pET29b-vip3-12811和pET29b-vip3-HD1)蛋白質之SDS-PAGE分析圖....................................61
圖13. 以親合性管柱純化重組菌株E. coli BL21 (pET29b-vip3-12811和pET29b-vip3-HD1)蛋白質之西方墨點分析圖.......................................62


表 目 錄

表1. 用於PCR之vip3基因引子對設計..............................................................63
表2. 重組蛋白Vip3 (pET29b-vip3-12811)與其他Vip3A蛋白胺基酸序列比較...............................................................................................................64
表3. 野生株仙人掌桿菌Bacillus cereus BCRC12811及蘇力菌B. thuringiensis kurstaki HD-1對不同齡期鱗翅目幼蟲之LC50 (μg/mL)..........................65
表4. 重組蛋白Vip3(pET29b-vip3-12811和pET29b-vip3-HD1)對不同齡期鱗翅目幼蟲之LC50 (μg/mL).........................................................................66

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